ESCRT disruption provides evidence against signaling functions for synaptic exosomes

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Abstract

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo Evenness Interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

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    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary: In this paper, Dresselhaus et al (2023) investigate the possibility that known cargoes of extracellular vesicles (EVs) released at the Drosophila neuromuscular junction have cell-autonomous functions rather than functions specifically conferred as a condition of their release in EVs, in vivo. To do so, authors focus their studies on use of Tsg101-KD, a mutant of the ESCRT-I machinery, of the ESCRT EV biogenesis pathway, and are able to show that for some endogenously-expressed, fluorescently-tagged cargoes, fluorescence intensity in the pre-synaptic compartment is significantly …

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    Referee #3

    Evidence, reproducibility and clarity

    Dresselhaus et al. investigates signaling functions for synaptic exosomes at the Drosophila NMJ. Exosomes are widely seen in vivo and in vitro. They are clearly sufficient to induce signaling responses in vitro, but whether they normally fulfill signaling functions in vivo has not been rigorously addressed. The authors make use of several mutants that block exosome release to test whether exosome release is important for two distinct signaling pathways: the Evi/Wg pathway and the Syt4 signaling pathway. Both pathways have been implicated in neuron to muscle signaling. Surprisingly, the authors find scant evidence that exosome …

  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript addresses the role of exosome secretion in neuromuscular junction development in Drosophila, a system that has been proposed to depend on exosomes. In particular, delivery of Wingless via exosomes has been proposed to promote structural organization of the synapse. Previously, however, the studies that proposed this model targeted the cargoes themselves, rather than targeting exosome biogenesis or secretion. In this new study, exosome biogenesis is targeted via knockdown of the ESCRT components Hrs, TSG101, and Chmp4. The authors find that some previously ascribed functions are not inhibited by these knockdowns. …

  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this paper, Dresselhaus et al (2023) investigate the possibility that known cargoes of extracellular vesicles (EVs) released at the Drosophila neuromuscular junction have cell-autonomous functions rather than functions specifically conferred as a condition of their release in EVs, in vivo. To do so, authors focus their studies on use of Tsg101-KD, a mutant of the ESCRT-I machinery, of the ESCRT EV biogenesis pathway, and are able to show that for some endogenously-expressed, fluorescently-tagged cargoes, fluorescence intensity in the pre-synaptic compartment is significantly elevated (Syt4 and Evi) and the postsynaptic …